. 2021 Mar 2;6(57):eabf7570.

doi: 10.1126/sciimmunol.abf7570.

Deep immune profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19

Laura A Vella^{1

2}, Josephine R Giles^{2

3

4}, Amy E Baxter^{2

3}, Derek A Oldridge^{2

5

6}, Caroline Diorio^{7

8}, Leticia Kuri-Cervantes^{2

9}, Cécile Alanio^{2

3

4}, M Betina Pampena^{2

9}, Jennifer E Wu^{2

3

4}, Zeyu Chen^{2

3}, Yinghui Jane Huang^{2

3}, Elizabeth M Anderson^{2

9}, Sigrid Gouma^{2

9}, Kevin O McNerney^{7

8}, Julie Chase^{7

10}, Chakkapong Burudpakdee⁷, Jessica H Lee⁷, Sokratis A Apostolidis^{2

11}, Alexander C Huang^{2

12}, Divij Mathew^{2

3}, Oliva Kuthuru^{2

3}, Eileen C Goodwin^{2

9}, Madison E Weirick^{2

9}, Marcus J Bolton^{2

9}, Claudia P Arevalo^{2

9}, Andre Ramos^{2

3}, C J Jasen¹³, Peyton E Conrey¹³, Samir Sayed¹³, Heather M Giannini¹⁴, Kurt D'Andrea¹⁵; UPenn COVID Processing Unit; Nuala J Meyer¹⁴, Edward M Behrens^{2

7

10}, Hamid Bassiri^{16

2

7}, Scott E Hensley^{2

9}, Sarah E Henrickson^{2

7

9

13}, David T Teachey^{7

8}, Michael R Betts^{2

9}, E John Wherry^{17

3

4}

Collaborators, Affiliations

Collaborators

Affiliations

¹ Division of Infectious Diseases, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA. vellal@chop.edu wherry@pennmedicine.upenn.edu.
² Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
³ Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
⁴ Parker Institute for Cancer Immunotherapy at University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
⁵ Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
⁶ Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
⁷ Immune Dysregulation Frontier Program, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
⁸ Division of Oncology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
⁹ Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
¹⁰ Division of Rheumatology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
¹¹ Division of Rheumatology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
¹² Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
¹³ Division of Allergy and Immunology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA,19104, USA.
¹⁴ Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
¹⁵ Division of Translational Medicine and Human Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
¹⁶ Division of Infectious Diseases, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
¹⁷ Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA. vellal@chop.edu wherry@pennmedicine.upenn.edu.

PMID: 33653907
PMCID: PMC8128303
DOI: 10.1126/sciimmunol.abf7570

Deep immune profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19

Laura A Vella et al. Sci Immunol. 2021.

. 2021 Mar 2;6(57):eabf7570.

doi: 10.1126/sciimmunol.abf7570.

Authors

Collaborators

Affiliations

¹ Division of Infectious Diseases, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA. vellal@chop.edu wherry@pennmedicine.upenn.edu.
² Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
³ Department of Systems Pharmacology and Translational Therapeutics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
⁴ Parker Institute for Cancer Immunotherapy at University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
⁵ Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA.
⁶ Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
⁷ Immune Dysregulation Frontier Program, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
⁸ Division of Oncology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
⁹ Department of Microbiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
¹⁰ Division of Rheumatology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
¹¹ Division of Rheumatology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
¹² Division of Hematology and Oncology, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
¹³ Division of Allergy and Immunology, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA,19104, USA.
¹⁴ Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104, USA.
¹⁵ Division of Translational Medicine and Human Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
¹⁶ Division of Infectious Diseases, Department of Pediatrics, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA.
¹⁷ Institute for Immunology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 19104, USA. vellal@chop.edu wherry@pennmedicine.upenn.edu.

PMID: 33653907
PMCID: PMC8128303
DOI: 10.1126/sciimmunol.abf7570

Abstract

Pediatric COVID-19 following SARS-CoV-2 infection is associated with fewer hospitalizations and often milder disease than in adults. A subset of children, however, present with Multisystem Inflammatory Syndrome in Children (MIS-C) that can lead to vascular complications and shock, but rarely death. The immune features of MIS-C compared to pediatric COVID-19 or adult disease remain poorly understood. We analyzed peripheral blood immune responses in hospitalized SARS-CoV-2 infected pediatric patients (pediatric COVID-19) and patients with MIS-C. MIS-C patients had patterns of T cell-biased lymphopenia and T cell activation similar to severely ill adults, and all patients with MIS-C had SARS-CoV-2 spike-specific antibodies at admission. A distinct feature of MIS-C patients was robust activation of vascular patrolling CX3CR1+ CD8+ T cells that correlated with the use of vasoactive medication. Finally, whereas pediatric COVID-19 patients with acute respiratory distress syndrome (ARDS) had sustained immune activation, MIS-C patients displayed clinical improvement over time, concomitant with decreasing immune activation. Thus, non-MIS-C versus MIS-C SARS-CoV-2 associated illnesses are characterized by divergent immune signatures that are temporally distinct from one another and implicate CD8+ T cells in the clinical presentation and trajectory of MIS-C.

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Figures

**Fig. 1. Cytopenias in MIS-C include enhanced T cell lymphopenia.**
(A) Overview of study cohorts. (B) Maximum clinical inflammatory markers. (C) Spearman correlation and hierarchical clustering of indicated clinical features and maximum laboratory values. Solid black boxes indicate mutually exclusive comparisons. (D) Spearman correlation of clinical parameters for which the lowest recorded (nadir) values are relevant for indicated features. (E) Nadir absolute lymphocyte counts (ALC). (F) Frequencies of total NK cells and NK subsets across adult healthy donors (HD), adult COVID-19, and pediatric cohorts. (G) Frequencies of ILCs (left) and CD8+ MAIT cells (right) across all cohorts. (H) Quantification of major lymphocyte populations across all cohorts. (**BEFGH**) Each dot represents an individual patient or subject, with adult HD in gray circles, adult COVID-19 in shades of mauve indicated by disease severity score, Ped COVID-19 in blue circles, with ARDS patients in dark blue, and MIS-C in green triangles. See key, top right. (BE) Normal laboratory reference ranges for healthy pediatric subjects are indicated in gray shading. Significance determined by unpaired Wilcoxon test between two pediatric groups for all clinical measures. P value shown when p <0.1, but >0.05. Lack of notation indicates no statistical significance. (CD) Spearman's Rank Correlation coefficient (ρ) is indicated by square size and heat scale; significance indicated by: * p<0.05, ** p<0.01, and *** p<0.001; black box indicates FDR<0.05 following Benjamini-Hochberg correction. See key, top right. (**FGH**) Significance determined by unpaired Wilcoxon test between adult HD and each pediatric group, and between pediatric groups, with adjustment for multiple comparisons using Benjamini-Hochberg correction, indicated by: * p<0.05, ** p<0.01, and *** p<0.001. Lack of notation for specified comparisons indicates no statistical significance. See Table S5 for subject numbers per panel.

**Fig. 2. T cell activation in MIS-C was greater than in COVID-19.**
(A) Proportion of naive cells among CD4+ (left) and CD8+ (right) T cells in all cohorts. (B) Association between age and proportion of naive T cells for pediatric cohorts and adult HD. (C) Representative plots and quantification of HLA-DR+CD38+ non-naive (nn)CD4+ T cells. (D) Representative plots and quantification of HLA-DR+CD38+ non-naive (nn)CD8+ T cells. (EF) Correlation between Ki67+ and HLA-DR+CD38+ populations for nnCD4+ (E) and nnCD8+ (F) T cells in pediatric cohorts. (G) Spearman correlation for indicated clinical features with frequencies of HLA-DR+CD38+ and Ki67+ nnCD4+ and nnCD8+ T cells. Top and middle: correlations within each cohort (Ped COVID-19 and MIS-C). Bottom: difference in Spearman correlation between the two pediatric cohorts. (**A-F**) Each dot represents an individual patient or subject. See key, top right. (A) Significance determined by unpaired Wilcoxon test between adult HD and each pediatric cohort, and between pediatric groups, with adjustment for multiple comparisons using Benjamini-Hochberg correction, indicated by: * p<0.05, ** p<0.01, *** p<0.001. Lack of notation for specified comparisons indicates no statistical significance. (CD) Significance determined by unpaired Wilcoxon test between adult COVID Severity Score 2 and each pediatric group, and between pediatric groups, with adjustment for multiple comparisons using Benjamini-Hochberg correction, indicated by: * p<0.05, *** p<0.001. Lack of notation for specified comparisons indicates no statistical significance. (EF) Non-parametric trend lines (Theil-Sen) for each pediatric cohort shown, with Spearman’s Rank Correlation coefficient (ρ) and P value. (G) Spearman's Rank Correlation coefficient (ρ) indicated by square size and heat scale; (bottom) the difference in ρ between Ped COVID-19 and MIS-C, divided by 2 to normalize to a -1 to 1 scale; permutation test significance indicated by: * p<0.05, ** p<0.01. Absence of black box indicates failure to meet FDR<0.05 following Benjamini-Hochberg correction. See Table S5 for subject numbers per panel.

**Fig. 3. MIS-C was uniquely marked by activation in CD8+ T cell populations associated with persistent antigen and vascular surveillance.**
(AB) Representative plots and quantification of PD-1+ nnCD4+ T cells (A) and nnCD8+ T cells (B). (C) Frequencies of CD39+ nnCD8+ T cells. (D) Frequencies of CD39+PD-1+ nnCD8+ T cells. (EF) Representative plots and quantification of CX3CR1+ nnCD8+ (E) and nnCD4+ T cells (F). (G) HLA-DR+CD38+ frequencies within CX3CR1- and CX3CR1+ nnCD4+ T cells. (H) Representative plots and quantification of HLADR+CD38+ population within CX3CR1- and CX3CR1+ nnCD8+ T cells. (IJ) Frequency of HLA-DR+CD38+ CX3CR1+ nnCD8+ T cells versus maximum D-Dimer (I) and platelet nadir (J). (K) Frequencies of HLA-DR+CD38+ CX3CR1+ (left) and CX3CR1- (right) nnCD8+ T cells in pediatric patients categorized by treatment with vasoactive medications. (L) Frequencies of HLA-DR+CD38+ CX3CR1+ (left) and CX3CR1- (right) nnCD8+ T cells in adult COVID-19 patients categorized by thrombotic complication. (**A-L**) Each dot represents an individual patient or subject. See key, top right. (**A-C, E-F**) Significance determined by unpaired Wilcoxon test between Ped COVID-19 and MIS-C groups only, indicated by: * p<0.05, ** p<0.01. (D) Significance determined between each pediatric group and adult COVID Severity Score 2 with adjustment for multiple comparisons using Benjamini-Hochberg correction, indicated by: * p<0.05 or P value shown where p >0.05 but <0.1. (GH) Significance determined by unpaired Wilcoxon test between Ped COVID-19 and MIS-C groups; or by paired Wilcoxon test between CX3CR1- and CX3CR1+ within pediatric groups, with adjustment for multiple comparisons using Benjamini-Hochberg correction, indicated by: * p<0.05, ** p<0.01, *** p<0.001. (IJ) Non-parametric trend lines (Theil-Sen) for total pediatric cohort (black), MIS-C (green) and pedCOVID-19 (blue), with Spearman’s Rank Correlation coefficient (ρ) and P value. (KL) Significance determined by unpaired Wilcoxon test between clinical category, indicated by: * p<0.05 or P value shown where p >0.05 but <0.1. Lack of notation for specified comparisons indicates no statistical significance. See Table S5 for subject numbers per panel.

**Fig. 4. Prolonged plasmablast responses in MIS-C.**
(A) Representative plots and quantification of plasmablasts (PB). (B) Frequency of PB within non-naive (nn) B cells versus anti-SARS-CoV-2 IgM (left) and IgG (right). (C) Representative histogram of T-bet expression by PB for an adult HD (gray), Ped COVID-19 (blue) and MIS-C (green) and quantification of T-bet+ PB frequencies. (D) SARS-Cov-2 antibody levels and (E) B cell features over days since admission in Ped COVID-19 (left) and MIS-C subjects (right). Black lines connect repeat draws for individual subjects. (F) Overview of adult recovered donor (RD) cohort. (G) Anti-SARS-CoV-2 IgM (left) and IgG (right) versus days since symptom start for RD cohort. (H) Frequency of PB within nnB cells versus days since symptom start for RD cohort. (**A-E, G-H**) Each dot represents an individual patient or subject, with adult HD in gray circles, adult COVID-19 in shades of mauve indicated by disease severity score, RD in purple circles, Ped COVID-19 in blue circles, with ARDS patients in dark blue, and MIS-C in green triangles. See key, top right. (A) Significance determined by unpaired Wilcoxon test between adult HD and each pediatric cohort, and between pediatric groups, with adjustment for multiple comparisons using Benjamini-Hochberg correction; indicated by **** p<0.0001. Lack of notation for specified comparisons indicates no statistical significance. (B) Non-parametric trend lines (Theil-Sen) for each pediatric cohort, with Spearman’s Rank Correlation coefficient (ρ) and P value. (C) Significance determined by unpaired Wilcoxon test between pediatric cohorts. (DE) For MIS-C, paired t test P value is shown for the three subjects with repeat draws. (EH) Gray shading indicates the derived central 95% adult HD reference interval. (G) Non-parametric trend lines (Theil-Sen) with Spearman’s Rank Correlation coefficient (ρ) and P value. Dotted gray line denotes positive assay threshold. See Table S5 for subject numbers per panel.

**Fig. 5. Immune perturbations in MIS-C overlapped with severe adult COVID-19 and corrected with clinical improvement.**
**(A)** Transformed UMAP projection of aggregated flow cytometry data from PBMC analysis. (B) Trajectory in UMAP space for repeat Ped COVID-19 ARDS draws. (C) Trajectory in UMAP space for repeat MIS-C draws. (D) Activated and proliferating CD8+ T cell populations over days since admission in Ped COVID-19 (left) and MIS-C subjects (right). (E) Flow cytometry plots for HLA-DR+CD38+ nnCD8+ for MIS-C patients with repeat draws. (F) Flow cytometry plots for Ki67+ nnCD8+ for MIS-C patients, as in (E). (G) CX3CR1+ HLA-DR+CD38+ and Ki67+ CD8+ T cell populations over days since admission in Ped COVID-19 (left) and MIS-C subjects (right). (H) Lymphocyte and platelet counts over days since admission for MIS-C subjects. (I) Clinical inflammatory markers over days since admission for MIS-C subjects. (**A-D,G-I**) Each dot represents an individual patient or subject. See key, top right. (**DGHI**) Black lines connect repeat draws for individual subjects. For MIS-C, paired t test P value is shown for the three subjects with repeat draws. (DG) Gray shading indicates the derived central 95% adult HD reference interval. (HI) Gray shading indicates normal clinical laboratory reference ranges for pediatric subjects. See Table S5 for subject numbers per panel.

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Update of

Deep Immune Profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19.
Vella L, Giles JR, Baxter AE, Oldridge DA, Diorio C, Kuri-Cervantes L, Alanio C, Pampena MB, Wu JE, Chen Z, Huang YJ, Anderson EM, Gouma S, McNerney KO, Chase J, Burudpakdee C, Lee JH, Apostolidis SA, Huang AC, Mathew D, Kuthuru O, Goodwin EC, Weirick ME, Bolton MJ, Arevalo CP, Ramos A, Jasen C, Giannini HM, DAndrea K, Meyer NJ, Behrens EM, Bassiri H, Hensley SE, Henrickson SE, Teachey DT, Betts MR, Wherry EJ. Vella L, et al. medRxiv [Preprint]. 2020 Oct 6:2020.09.25.20201863. doi: 10.1101/2020.09.25.20201863. medRxiv. 2020. Update in: Sci Immunol. 2021 Mar 2;6(57):eabf7570. doi: 10.1126/sciimmunol.abf7570. PMID: 32995826 Free PMC article. Updated. Preprint.

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Deep immune profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19

Collaborators

Affiliations

Deep immune profiling of MIS-C demonstrates marked but transient immune activation compared to adult and pediatric COVID-19

Authors

Collaborators

Affiliations

Abstract

Figures

Update of

References

Publication types

MeSH terms

Supplementary concepts

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Research Materials

Miscellaneous